Figure 4

Kinetics of dissociation of beta/gamma from cyclase(s) complex formed during simultaneous activation of [GABA]b and β-adrenergic receptors (A, B) Plasma membranes (50 μg protein) were incubated with drugs (200 nM) and GTP (10 μM) + EDTA (1 mM) at 20°C for 20 min then placed in ice with Mg (5 mM) and an excess of GDP (1 mM) for 1, 2 or 5 min. Membranes were immediately solubilized (see 'materials and methods'). A 'pull down' experiment using Forskolin-agarose was performed in ice followed by gel electrophoresis, and Western blot analysis with anti-beta, of the material bound to the affinity beads. A: dissociation of G beta /cyclase complex: analysis of baclofen baclofen: at 1, 2, 5 min. A control t = 0, and a control t = 0 using membrane treated with pertussis toxin (PTx), are shown. B: dissociation of G beta /cyclase complex: analysis of the coincidence iso + bac Top: baclofen + isoproterenol: at 1, 2, 5, 10 min. Middle: baclofen + isoproterenol + GTPγS: membranes were incubated in presence of GTPγS (10 μM) and Mg (5 mM) for 20 min at 20°C, then placed in ice with an excess of GDP (1 mM) (t = 0). Membranes were then solubilized at the indicated times and a 'pull down' experiment was carried out as indicated above. Bottom: baclofen + isoproterenol + cholera toxin (CTx) treatment. Same experiment as above except membranes were incubated with GTP (10 μM) + cholera toxin (CTx) instead of GTPγS. The arrow corresponds to MW = 35 kDa.